The influence of microstructure on slip irreversibility and crack initiation during high-cycle fatigue of a titanium alloy has been explored. The results indicate that the fatigue strength of lamellar microstructure is higher than the bimodal microstructure. The underlying microstructure and strain partition below the fatigue crack origins have been revealed through electron backscattered diffraction (EBSD). Furthermore, the slip irreversibility and the corresponding accumulative strain to fracture were calculated and compared for these two microstructures. Finally, a critical parameter about (8.1 +/- 2) x 10(-4)center dot mu m(-2) for fatigue crack initiation was achieved with the present titanium alloy, which was found to be independent of microstructural types.